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Abstract

Appropriate diagnosis is the key factor for treatment of viral diseases. Time is the most important factor in rapidly developing and epidemiologically dangerous diseases, such as influenza, Ebola and SARS. Chronic viral diseases such as HIV-1 or HCV are asymptomatic or oligosymptomatic and the therapeutic success mainly depends on early detection of the infective agent. Over the last years, aptamer technology has been used in a wide range of diagnostic and therapeutic applications and, concretely, several strategies are currently being explored using aptamers against virus proteins. From a diagnostics point of view, aptamers are being designed as a bio-recognition element in diagnostic systems to detect viral proteins either in the blood (serum or plasma) or into infected cells. Another potential use of aptamers is for therapeutics of viral infections, interfering in the interaction between the virus and the host using aptamers targeting host-cell matrix receptors, or attacking the virus intracellularly, targeting proteins implicated in the viral replication cycle. In this paper, we review how aptamers working against viral proteins are discovered, with a focus on recent advances that improve the aptamers' properties as a real tool for viral infection detection and treatment.

Introduction

Viruses are infectious agents that enter and replicate only inside the living cells of other organisms. Once the virus replicates inside the cell, it may remain dormant for long periods of time or be released immediately and attach to other healthy cells to begin the infection process again. Many diseases are caused by viruses such as the influenza, hepatitis, human immunodeficiency virus (HIV) or emerging viral diseases. While they differ in symptoms such as fever and weakness, some present no symptoms at all. Rapid and secure diagnosis of viral infections is a key factor for treatment of these diseases avoiding new spread.

Aptamers to HIV Genome

(2) The outer envelope has gp120 and gp40 proteins involved in interaction with cellular receptors (CD4, CCR5, NLS) and fusion to cellular membrane; (3) In the cytoplasm the ssRNA viral genome is released and the retro-transcription step is produced; (4) The integrase protein binds to dsDNA viral genome by LTR ends sequences and other cellular proteins forming the pre-integration complex (PIC); (5) The PIC goes into nucleus through the nuclear pore and is integrated in the cellular genome by the integrase protein activity (provirus); (6) The viral RNAs are transcripted from proviral DNA and exported to cytoplasm to translate viral proteins as protease and a big pre-protein that are assembled to new RNA viral genomes and leave the cell with outer envelope from the cellular membrane; (7) After the budding viral particle, the proteases process the big pre-protein and get a mature viral particle.

Aptamers to HIV as Antiviral Agents

At present, the treatment HIV-1/AIDS is by a combination of several antiretroviral drugs (cART), which can slow the progress of the disease and reduce the risk of death and disease complication, but it is not curative. Moreover, many patients do not tolerate cART because it has severe side effects, and it is too expensive for patients in developing countries. In this regard, aptamers have been considered an alternative or adjuvant to the chemical antiviral agents in cART to overcome these limitations. To date, highly specific, nucleic acid-based aptamers that target various parts of HIV-1 genomes, HIV-1 proteins (including HIV-1 protease (PR), reverse transcriptase (RT), nucleocapsid, gp120, and Gag) and cellular proteins (nucleolin, CD4 or CCR5) have been isolated and shown to effectively suppress viral replication to apply in HIV therapy [5, [25] [26] [27] (Figure 2 ). (1) The HIV viral particle has an inner capside containing ssRNA viral genome and integrase and retro-transcriptase proteins, mainly; (2) The outer envelope has gp120 and gp40 proteins involved in interaction with cellular receptors (CD4, CCR5, NLS) and fusion to cellular membrane; (3) In the cytoplasm the ssRNA viral genome is released and the retro-transcription step is produced; (4) The integrase protein binds to dsDNA viral genome by LTR ends sequences and other cellular proteins forming the pre-integration complex (PIC); (5) The PIC goes into nucleus through the nuclear pore and is integrated in the cellular genome by the integrase protein activity (provirus); (6) The viral RNAs are transcripted from proviral DNA and exported to cytoplasm to translate viral proteins as protease and a big pre-protein that are assembled to new RNA viral genomes and leave the cell with outer envelope from the cellular membrane; (7) After the budding viral particle, the proteases process the big pre-protein and get a mature viral particle.

Use of Aptamers for Delivery of Therapeutic Molecules

Aptamers can also serve as elements that selectively recognize and bind to defined cell types or tissues. By attaching drug molecules, the aptamers can be used to deliver cargo molecules to or into specific cells or tissues of interest [64] . In order to reach efficient RNAi activity, aptamer-siRNA conjugates must be successfully internalized and released into the cytoplasm where they can meet the RNAi machinery [65] . To improve the Dicer entry and processing of the siRNA, one pair of complementary guanosine and cytosine (GC)-rich "sticky bridge" sequences can be chemically appended to the 3 end of the aptamer and one of the siRNA strands, respectively. Both the aptamer and siRNA portions are chemically synthesized and subsequently annealed via "sticky bridge" [66] .

Aptamers to Structural Proteins E1, E2 and Core

Finally, an innovative approach to inhibiting HCV viral infection has been designed based on sequestration of miR-122, implicated as a regulator of fatty-acid metabolism in mammals, exclusively in infected cells. To this end. Lee et al. have generated a chimeric molecule composed by a hammerhead ribozyme conjugated with an aptamer against NS5B protein. After aptamer recognition of HCV infection, ribozyme domain released a sequence complementary to miR-122 producing the inhibition of HCV replication in Huh-7 cells [118] .

Introduction

Viruses cause different types of damage to the body which, if left untreated, can lead to death. There are many antiviral drugs that block the infection process at different stages. Some drugs prevent the virus from interacting with the healthy cell by blocking a receptor that helps internalize the virus into the cell. Other drugs inhibit the proliferation of the virus within the cell. The simultaneously use of several drugs affecting different processes increases the probability of recovery of the patient. Although some viral infections such as hepatitis or HIV remain latent for a long time current treatments control the virus and prevent further damage to the body. Viral infections usually produce an immune response in the host that eliminates the infecting virus. The same protective effect is produced by vaccines, which confer an artificially acquired immunity to the viral infection. However, some viruses including those that cause acquired immune deficiency syndrome (AIDS) and viral hepatitis evade these immune responses and result in chronic infections. Antibiotics have no effect on viruses, but several antiviral drugs have been developed. Because viruses use vital metabolic pathways within host cells to replicate, they are difficult to eliminate without using drugs that cause toxic effects to host cells in general. The most effective medical approaches to viral diseases are vaccinations to provide immunity to infection, and antiviral drugs that selectively interfere with viral replication.
Most of the antiviral drugs are nucleoside analogues which lack the hydroxyl groups. Viruses mistakenly incorporate these analogues into their genomes during replication and, in consequence, the newly synthesized DNA is inactive and the life-cycle of the virus is then halted. Some of the most frequently prescribed antiviral nucleoside analogues based-drugs are aciclovir for Herpes simplex virus infections and lamivudine for HIV and Hepatitis B virus infections [1] . During the last years, the nucleoside analogue drug ribavirin combined with interferon has been used for hepatitis C treatment [2] , although currently there is a more effective treatment that includes simeprevir available for patients with genotype 1 and genotype 4 [3] . The treatment of chronic carriers of the hepatitis B virus by means of a similar strategy using lamivudine has been developed [4] . Today, the first line treatment of choice includes one of three drugs: Peg-IFN, entecavir or tenofovir because of their greater power and because they produce a very low rate of resistance.

Aptamers against Human Immunodeficiency Virus (HIV)

Human immunodeficiency virus is a lentivirus (a subgroup of retrovirus) that causes HIV infection and over time AIDS [21] . HIV infects essential cells in the human immune system such as CD4 + T cells, macrophages, and dendritic cells. When CD4 + T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections. HIV enters cells by endocytosis through the interaction of gp120 viral surface protein (SU) with CD4 host cell. The subsequent interaction of this complex with chemokine coreceptors produces a conformational change in viral protein gp41 that promotes the fusion of virion and target cell membranes leading to the release of HIV particles into the cell. Once inside the cell, viral uncoating generates the viral reverse transcription complex, and the reverse transcription gives the HIV preintegration complex (PIC). The PIC gets into the nucleus and the HIV DNA (provirus) is integrated into the cellular chromosome. Integration can lead to either latent or transcriptionally active forms of infection. The latent form gives the viral latency in cells that can replicate in new infected cells; this provirus can remain hidden during years or replicate and form new viral particles in any moment. The transcriptional active forms are transcript and translated forming new viral particles that dead the host cell and goes to infect new cells. Great efforts are being made to get sensitive, fast and simple diagnostic methods and effective therapies.

Aptamers to Nucleocapsid Protein

Aptamers to Surface Glycoprotein (gp 120) Gp120 is essential for virus entry into cells as it plays a vital role in attachment to specific cell surface receptors mainly on helper T-cells. Several small aptamers containing G-quadruplex selected against gp120 have demonstrated antiviral activity [33] . The first of these molecules was the phosphorothioate 8-mer d(TTGGGGTT), named ISIS 5320, which forms a tetrameric G-quadruplex structure that binds the V3 loop of gp120 inhibiting virus entry [48] . Later on, Koizumi et al. synthesized a set of G-rich oligonucleotides and identified the hexadeosyribonucleotide d(TGGGAG), known as Hotoda's sequence, [49, 50] . Several authors have used the Hotoda's sequence as a lead sequence to make a series of modifications at the 5 and 3 ends of the molecule or mutations in the sequence that allowed to find molecules with high anti-HIV activity [50] [51] [52] [53] [54] [55] [56] .

Aptamers against HBV

Hepatitis B virus (HBV) is a partially double-stranded DNA virus of the Hepadnaviridae family classified into eight genotypes from A to H. The main element of the viral particle of HBV virus and also the most characterized component is the hepatitis B surface antigen (HBsAg) [72] .

Aptamers to 5′ and 3′ Untranslated Regions (5′ and 3′UTR)

Non-translated 5′ and 3′ regions have highly conserved sequences and structured regions closely related with transcription and replication of the HCV virus. Specifically, 5′ end contains the Internal

Aptamers to 5 and 3 Untranslated Regions (5 and 3 UTR)

Non-translated 5 and 3 regions have highly conserved sequences and structured regions closely related with transcription and replication of the HCV virus. Specifically, 5 end contains the Internal Ribosome Entry Site (IRES) domain responsible of transcription initiation by ribosome recognition of HCV viral genome. However, 3 UTR includes a region essential for viral replication named cis-acting replication element (CRE).

Aptamers to Nonstructural Protein 3 (NS3)

Multifunctional enzyme NS3 is an essential protein for virus survival and it is considered a good target for the development of new antiviral-drugs. The protease activity of the protein is found in the N-terminal domain and the helicase activity is present in the C-terminal domain of the enzyme. During the last years, the group of Nishikawa has been working with different sets of RNA aptamers against the full-length or truncated NS3 protein to inhibit its dual-activity. Initially, they isolated NS3-specific aptamers that inhibited the protease activity (10-G1) or the dual activity, protease/helicase, of the enzyme NS3 (G6-16 and G6-19) [101, 102] . Afterward, new RNA aptamers against the helicase domain of NS3 protein, named G9-I, II and III, were able to inhibit protease activity in vitro [103] . Later on, the authors described the interaction between G9-I aptamer with Arg161/Arg130 residues in the truncated NS3 form as a putative target for protease activity inhibition [104] . To stabilize and protect G9 aptamers against exonuclease activity in vivo, Nishikawa et al. conjugated G9-II aptamer to the stem IV region of the Hepatitis delta virus (HDV) ribozyme. In addition, to allow nuclear export of the aptamer, the chimeric molecule HDV-G9-II (HA) was fused to a constitutive transport element (CTE) generating HAC molecule. Finally, the protease-inhibition capability of G9-II aptamer was checked, using HA and HAC expression vector in vivo [105, 106] . In order to inhibit the dual activity of NS3, a poly U tail in the 5 or 3 ends of G9-I aptamer (5 -14U-NEO-III or NEO-III-14U) was added. The two constructions were able to inhibit with high efficiency of the protease and helicase activities of NS3. Moreover, NEO-III-14U decreased the interaction between NS3 protein and the 3 end of the positive or negative sense HCV RNA and inhibited protease activity of NS3 in vivo [107] . Next, a new set of RNA aptamers were selected against the helicase domain using the truncated NS3 protein (NS3h) and the aptamer with greatest capability to deplete helicase activity in vitro was identified and named aptamer #5 [108] . Finally, a dual-functional aptamer named G925-s50 was designed using a truncated version of aptamer #5 plus G9-II aptamer linked by 50 mer poly(U) spacer. The designed molecule G925-s50 showed a significant inhibition of NS3 helicase-protease activity in vivo and is proposed by Nishikawa group as the best candidate for anti-HCV therapy [109] .

Aptamers against Human Papilloma Virus (HPV)

Human papillomavirus (HPV) is a DNA virus from the papillomavirus family. Most HPV infections cause no symptoms and resolve spontaneously, but some of them persist and result in warts or precancerous lesions which increase the risk of cancer of the cervix, vulva, vagina, penis, anus, mouth, or throat [119, 120] .

Aptamers to HPV in Diagnostics

Several proteins from human papillomavirus, particularly E6 and E7, promote tumor growth and malignant transformation and are frequently associated with cervical cancer. Thus, these proteins represent ideal targets for diagnostic and therapeutic strategies. Belyaeva et al. reported two RNA aptamers to E6, named F2 and F4, which induced apoptosis in cells derived from an HPV16-transformed cervical carcinoma. This aptamers were able to inhibit the interaction between E6 and PDZ1 from Magi1, with F2 being the most effective inhibitor, while none of them inhibited E6-p53 interaction or p53 degradation [121] .
Toscano-Garibay et al. isolated an aptamer (G5α3N.4) that exhibited specificity for E7 with a Kd comparable to aptamers directed to other small targets [122] that may be used for the detection of papillomavirus infection and cervical cancer. The same group characterized an RNA aptamer, named Sc5-c3, that recognized baculovirus-produced HPV-16 L1 virus-like particles (VLPs) with high specificity and affinity (Kd = 0.05 pM). This aptamer produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection [123] . Recently, Graham and Zarbl identified several DNA aptamers that have high affinity and specificity to the non-tumorigenic, revertant of HPV-transformed cervical cancer cells, which can be used to identify new biomarkers that are related to carcinogenesis produced by HPV [124] .

Aptamers to Herpes Simplex Virus (HSV)

Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) are two members of the herpesvirus family, Herpesviridae, that infect epithelial tissues before invading the nervous system, where it becomes latent. Unfortunately until now, it has not found any treatment to eradicate the virus [128] . Table 5 shows information on the aptamers described against HPV and HSV. Aptamer technology has been used by Corbin-Lickfett et al. to identify RNA sequences capable of being recognized by HSV-1 ICP27 protein, an important regulator for viral gene expression. After SELEX procedure, GC-rich RNA sequences were isolated, which did not form stable secondary structures [129] . With a therapeutic purpose, Gopinath et al isolated two RNA aptamers (aptamer-1 and aptamer-5) against the ectodomain of the gD protein of HSV-1, which plays an important role in viral entry to the host cells. These aptamers specifically bind to gD protein of HSV-1 with high affinity but not the gD protein of HSV-2. Furthermore, aptamer-1 efficiently blocked the interaction between the gD protein and the HSV-1 target cell receptor (HVEM) in a dose-dependent manner with a EC 50 in the nanomolar range. Anti-HSV-1 activity of aptamer-1 was analyzed by using plaque assays and the results showed that this aptamer efficiently inhibited viral entry. A shorter variant of aptamer-1 named mini-1 aptamer (44-mer) had at least as high an affinity, specificity, and ability to interfere with gD-HVEM interactions [130] . In a similar way, Moore et al. have reported the isolation and characterization of one aptamer, G7a, that binds the gD protein of HSV-2 and neutralizes infection through the Nectin1 and HVEM entry receptors with IC 50 of 20 nM [131] . Interestingly, aptamers that prevent HSV-2 infection may also reduce the morbidity associated with HIV-1 as HSV-2 is a major risk factor for the acquisition of HIV-1.

Aptamers to Influenza

Influenza is considered the most prevalent infectious disease in humans. Three emerging influenza viruses were responsible for major pandemics in the twentieth century: the 1918 Spanish influenza virus, the 1957 Asian influenza virus, and the 1968 Hong Kong influenza virus [132] . Indeed, the 1918 Spanish influenza virus was estimated to have killed 20-50 million people worldwide [133] . More recently, a highly pathogenic avian virus of the H5N1 subtype has produced sporadic infections in humans and, while it is associated with high rates of mortality, its poor transmission in humans prevented a more extensive spread among human populations. However, in 2009, a new influenza A virus of the H1N1 subtype emerged (pH1N1) that possessed high transmissibility but relatively low virulence, rapidly spreading across the entire globe and causing the first pandemic of the 21st century [134, 135] . Subsequently, 2013 witnessed the appearance of a new highly pathogenic avian virus of the H7N9 subtype in China [136] .
Influenza viruses are enveloped RNA virus of the family Orthomyxoviridae. The virion surface carries two membrane glycoprotein components, hemagglutinin (HA) and neuraminidase (NA) and, in the central core, the viral RNA (negative-sense) genome fragmented into eight single-stranded molecules and viral proteins that package and protects this RNA. Each segment contains one or two genes that code for the 15 viral proteins. Highly variable surface proteins, HA and NA, are used to classify influenza subtypes. The combination of hemagglutinin and neuraminidase mainly determines the host organism and the viral infectiousness. Currently, 18 HA and 11 NA types have been identified being the subtypes H1, H2 and H3, and N1 and N2 commonly found in humans.

Aptamers to Influenza Virus in Diagnostics

The detection rapid of influenza virus as well as the categorization of these viruses is particularly important due to the high risk of infection, the rapid propagation and the high frequency of mutation that often induces the arrival of new strains that can cause epidemics and even pandemics. An extensive review about the diagnostic strategies for influenza has been recently published [137] .
The recent advances in the development of rapid, automatic, point of care devices for the diagnosis and subtyping of influenza virus are sustained in two facts: (i) the rapid spread of influenza-associated H1N1 viruses that has caused serious concern in recent years; and (ii) H5N1 subtype of the avian influenza virus (AIV) caused the most lethal outbreaks of highly pathogenic avian influenza (HPAI) in poultry and fatal infections in human cases for over a decade. Thus, aptamers have been generated and found to be specific against these recent pandemic influenza viruses A/H1N1pdm [142] and H5N1 [143] .
Lee et al. developed an integrated microfluidic system that was used to screen a specific aptamer for the influenza A/H1N1 virus in an automated and highly efficient manner [144] . The selected aptamer showed a specific and sensitive detection of the influenza A/H1N1 virus, even in biological samples such as throat swabs. Later, they used a new approach for fluorescence-based detection of the influenza A H1N1 virus using a sandwich-based aptamer assay that is automatically performed on an integrated microfluidic system [145] . The entire detection process was shortened to 30 min using this chip-based system which is much faster than the conventional viral culture method. The limit of detection was significantly improved due to the high affinity and high specificity of the H1N1-specific aptamers. In addition, this two-aptamer microfluidic system had about 10 3 times higher sensitivity than the conventional serological diagnosis. The conformation of the aptamers changes in response to the solvent composition, including ion type and concentration, pH, and temperature. On the basis of this, Wang et al. have developed a microfluidic system that exploited the predictable change in conformation of the aptamer previously used in the group, exposed to different ion concentrations in order to detect multiple types of the influenza virus [146] . Thus, a single fluorescent-labelled aptamer is able to identify three different influenza viruses (influenza A H1N1, H3N2, and influenza B) at the same time, by modifying operating conditions, in 20 min. This chip-based aptamer-binding assay has several important advantages; it is rapid, accurate, and cheaper than multiple-aptamer screening.
Current methods for H5N1 AIV detection are virus isolation and RT-PCR that requires several days and expensive equipment and reagents. Rapid detection assays are also available (such as ELISA or immunochromatographic strips) but are less sensitive and specific. The alternative approach is biosensors technology, several biosensors have been developed to detect AIV among them biosensors using as probe aptamers (aptasensors) (reviewed in [147] . In the Li's lab, a highly specific DNA aptamer that can bind H5N1 virus with high affinity was selected. Using this aptamer, other authors have developed different aptasensors based on Surface Plasmon Resonance (SPR) [148] , a quartz crystal microbalance (QCM) aptasensor crosslinked polymer hydrogel [149] and several aptasensors based on impedance methods [150] [151] [152] . These aptasensors were able to detect H5N1 quickly and/or with more sensitivity than antibody-based biosensors.
The impedance-based aptasensor described Fu et al. has the lowest detection limit, however, it requires signal amplification with labels and a prolonged detection limit [150] . The impedance aptasensor with microfluidics chips has a lower detection limit than the SPR-based aptasensor [148] and the same sensitivity as the QCM aptasensor [149] , but the QCM-based aptasensors are not practical for in-field use due to the QCM's predisposition to environmental noise. The major advantage of the impedance aptasensor with gold nanoparticles for signal amplification described by Karash et al. is that it requires a small sample volume and is cheaper than the detection platforms based on QCM or that use interdigitated electrode microfluidic chips [152] . Recently, Nguyen et al. developed a sandwich-type SPR-based biosensor for the detection of H5Nx viruses using a pair of aptamers selected against a mixture of H5Nx whole viruses using Multi-GO SELEX [153] . The sensitivity of the dual aptamer-based system increased by more than 50-fold than for single-aptamers. In addition, the sensitivity was additionally enhanced when the secondary aptamer was conjugated with gold nanoparticles.

Aptamers to Influenza Virus as Antiviral Agents

Several aptamers against influenza virus have been developed for therapeutics purposes, mainly targeting hemagglutinin (reviewed in [5] ) (Figure 4) . These aptamers are able to inhibit the entry of the virus of the cells by blocking hemagglutinin activity. The common technique to measure the inhibitory activity of the aptamers in vitro is the hemagglutination inhibition assay. The model was more extensively used to test the effect of the aptamers on the viral infection involving the use of cell cultures, mainly Madin-Darby canine kidney (MDCK) cells. The cells are infected with the virus and incubated with the aptamers and the inhibition of viral infectivity is tested. Using these assays, DNA and RNA aptamers selected against HA from Influenza A virus [142, [154] [155] [156] [157] [158] or avian influenza virus [159] [160] [161] , able to significantly decrease the viral infection in cells, have been described. However, only a few studies have described aptamers capable of mediating a reduction in viral pathogenicity in mice models. Jeon et al. evaluated the effect of the administration intranasal of the A22 aptamer, a DNA aptamer selected against the HA-(91-261) peptide, in mice before, at the same time and after virus infection [154] . The aptamer-induced inhibition of viral infection was determined by prevention of weight loss, decrease of viral load in the lungs and restriction of the level of inflammation and cellular infiltration. A22 reduced up to 95% of infection in all the strains tested (H1N1, H2N2 and H3N2). A22 was most effective when administered concomitantly with the viral infection leading to 95% reduction in viral burden. The administration of A22 one day prior to infection (preventive treatment) was less effective, probably because the DNA is partially degraded. Interestingly, the treatment with A22 two days following the infection (therapeutic treatment) still leads to almost 95% reduction in viral titer in the lungs of the mice. In 2014, Musafia et al. used A22 aptamer as a starting point and the quantitative structure-activity relationship (QSAR) tool to produce aptamers with 10-15 times more potent antiviral activity in animal models than A22 aptamer. The binding of these aptamers to the virus (20 times higher than A22) may not necessarily be sequence-specific being the most important properties the aptamer length, 2D-loops and repeating sequences of C nucleotides [157] .
Another antiviral strategy is the inhibition of the enzymes involved in the viral replication, transcription and translation. The polymerase complex of Influenza virus catalyzes the viral replication and transcription. This heterotrimer is composed of three subunits named PA, PB1 and PB2 [164] [165] [166] . PA plays the role of an endonuclease, cleaving host mRNAs downstream of their mRNA cap structures, which are recognized and bound by PB2 [167] . The N-terminal of the PA subunit (PA N ), which holds the endonuclease activity site, is highly conserved among different subtypes of influenza virus, which suggests it is an attractive target in the development of anti-influenza agents. Yuan et al. selected DNA aptamers against both PA protein (three aptamers), and the PA N domain (six aptamers) of an H5N1 virus strain [168] . Four of the six PA N selected aptamers inhibited both endonuclease activity and H5N1 virus infection whereas the three PA-selected aptamers did not inhibit endonuclease activity and virus infection. Finally, one of the four effective aptamers, exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC 50 ) around 10 nM.
Pharmaceuticals 2016, 9, 78 19 of 33 aptamers, exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC50) around 10 nM. The association of the viral polymerase, bound to the cap, and eIF4GI may be involved in the preferential translation of viral mRNAs during influenza infection. In addition, the interaction of NS1, bound to a conserved 5untranslated region (UTR) element of the viral mRNA, with eIF4GI and PABP1 could promote the formation of a "closed loop" between the 5′ and 3′ ends of the viral mRNA; (4) RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/β production. Mitochondrial antiviral-signaling (MAVS) protein. Table 6 shows information on the aptamers described against influenza virus. Vaccination is a powerful approach to diminish the effects of influenza epidemics, but the use of antiviral drugs can also be very useful, particularly in delaying the spread of new pandemic viruses. Neuraminidase inhibitors like oseltamivir, laninamivir, zanamivir, and peramivir are commonly used as antiviral agents to treat influenza infection, especially in Japan. However, because of the rapid increases in drug-resistant influenza virus, it is essential to develop new antiviral drugs as an emerging strategy to block cellular factors important for the infective cycle. The advantage of blocking important cellular pathways for the virus inhibitory effect is that, in principle, it is not specific of influenza strain and the emergence of resistant virus is minimized. A limited number of aptamers targeting host cell factors have been described. Of these, the use of RIG-I as a target for aptamers to control viral infection should be emphasized [169] . RIG-I is a cytosolic receptor for nonself RNA that mediates immune responses against viral infections through IFNα/β production [170] . The use of a specific RIG-I aptamer that activates RIG-I efficiently blocks the replication of the Newcastle disease virus, vesicular stomatitis virus and influenza virus in infected cells, evidencing that aptamers targeting cellular factors can act as efficient antiviral agents [169] . The association of the viral polymerase, bound to the cap, and eIF4GI may be involved in the preferential translation of viral mRNAs during influenza infection. In addition, the interaction of NS1, bound to a conserved 5-untranslated region (UTR) element of the viral mRNA, with eIF4GI and PABP1 could promote the formation of a "closed loop" between the 5 and 3 ends of the viral mRNA; (4) RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/β production. Mitochondrial antiviral-signaling (MAVS) protein. Table 6 shows information on the aptamers described against influenza virus. Vaccination is a powerful approach to diminish the effects of influenza epidemics, but the use of antiviral drugs can also be very useful, particularly in delaying the spread of new pandemic viruses. Neuraminidase inhibitors like oseltamivir, laninamivir, zanamivir, and peramivir are commonly used as antiviral agents to treat influenza infection, especially in Japan. However, because of the rapid increases in drug-resistant influenza virus, it is essential to develop new antiviral drugs as an emerging strategy to block cellular factors important for the infective cycle. The advantage of blocking important cellular pathways for the virus inhibitory effect is that, in principle, it is not specific of influenza strain and the emergence of resistant virus is minimized. A limited number of aptamers targeting host cell factors have been described. Of these, the use of RIG-I as a target for aptamers to control viral infection should be emphasized [169] . RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/β production [170] . The use of a specific RIG-I aptamer that activates RIG-I efficiently blocks the replication of the Newcastle disease virus, vesicular stomatitis virus and influenza virus in infected cells, evidencing that aptamers targeting cellular factors can act as efficient antiviral agents [169] . However, aptamers directed against cellular factors that establish essential interactions with influenza virus proteins had not been reported before. The mRNAs of influenza virus possess a 5 cap structure and a 3 poly (A) tail that makes them structurally indistinguishable from cellular mRNAs. However, selective translation of viral mRNAs occurs in infected cells through a discriminatory mechanism, whereby viral polymerase and NS1 interact with components of the translation initiation complex, such as the eIF4GI and PABP1 proteins [171] [172] [173] . Thus, the inhibition of viral protein-translation factor interactions or their destabilization can be potentially used as an antiviral strategy. Recently, Rodriguez et al. studied whether two aptamers which bind hPABP1 with high affinity (ApPABP7 and ApPABP11) are able to act as anti-influenza drugs [174] . Both aptamers inhibit influenza virus replication of H1N1 or H3N2 subtypes at high and low multiplicity of infection and the viral polymerase-eIF4GI interaction. In addition, aptamer ApPABP11 inhibits the interactions between NS1 and eIF4GI or PABP1. These results indicate that aptamers targeting the host factors that interact with viral proteins may potentially have a broad therapeutic spectrum, reducing the appearance of escape mutants and resistant subtypes.

Aptamers against Other Emerging Viruses

An emergent virus is a virus that has adapted and emerged as a new disease/pathogenic strain, with attributes facilitating pathogenicity in a field not normally associated with that of virus. This includes viruses that are the cause of a disease which has notably increased in incidence; this is often a result of a wide variety of causes from both the influence of man and nature. Most emergent viruses can be categorized as zoonotic (an animal disease that can be transmitted to humans), and this has the advantage of possibly having several natural reservoirs for the disease.
Most of these viruses have newly appeared in a population or have existed but are rapidly increasing in incidence or geographic range and only recently aptamers against emergent viruses such as Rift Valley Fever, Tick-borne encephalitis, Dengue, Ebola viruses or other arboviruses have been developed [175] .

Aptamers to Rift Valley Fever Virus (RVFV)

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus (genus Phlebovirus) responsible for widespread outbreaks of severe disease such as hepatitis, encephalitis and hemorrhagic fever in humans [176] . The virus is endemic throughout much of the African continent. However, the emergence of RVFV in the Middle East, northern Egypt and the Comoros Archipelago has highlighted that the geographical range of RVFV may be increasing, and has led to the concern that an incursion into Europe may occur. At present, there is no licensed human vaccine [177] .
The nucleocapsid protein (N) of RVFV is an RNA binding protein required for the production of viable virus because of its involvement in several stages of viral replication. This protein protects the viral genome from degradation and prevents the formation of double stranded RNA intermediates during replication and transcription by encapsidating viral genomic and antigenomic RNA [178] . Ellenbecker et al. isolated RNA aptamers that bound N with high affinity and identified GAUU and pyrimidine/guanine motifs in their sequences, which are also present within the coding region of the RVFV genome. Furthermore, the authors developed a truncated RNA aptamer labeled with fluorescein using a fluorescence polarization (FP) system. Titration of N with the 3 -FAM-labeled RNA aptamer gave an apparent Kd of 2.6 µM. Competitive binding experiments were conducted with four different aptamers and the apparent Ki values were all in the~200 nM range. These data demonstrate that these aptamers might be used to construct a sensitive fluorescence based sensor of N binding with potential applications for drug screening and imaging methodologies [179] .

Aptamers to Tick-Borne Encephalitis Virus (TBEV)

Tick-borne encephalitis virus (TBEV) belongs to the family Flaviviridae, genus Flavivirus. This virus produces tick-borne encephalitis (TBE), an important emerging infectious disease that targets the central nervous system (CNS) [180] . There is currently no specific antiviral treatment for TBE because the specific immunoglobulin used in clinical practice has several disadvantages. The purpose of Kondratov et al. was to obtain an aptamer population against a fragment of the surface protein E of the TBEV, since it is available for aptamers outside of the host cell [181] . Authors showed that the treatment with the library of aptamers produced a TBEV neutralization index comparable with the results of neutralization of the commercial human immunoglobulin against tick-borne encephalitis (NPO Microgen, Russia). In addition, the enzyme immunoassay systems based on the immobilization of viral particles on antibodies are most commonly used for the TBEV diagnosis and the authors claim that protein E aptamers could substitute antibodies in these systems.

Aptamers to Dengue Virus (DENV)

Dengue viruses (DENVs) belong to the Flaviviridae family, and contain four serologically and genetically distinct viruses, termed DENV-1, DENV-2, DENV-3 and DENV-4. The envelope (E) protein plays an important role in viral infection but, however, there is no effective antibody for clinical treatment due to antibody dependent enhancement of infection. Chen et al. identified an aptamer (S15) that can bind to DENV-2 envelop protein domain III (ED3) with a high binding affinity. S15 aptamer was found to form a parallel quadruplex structure that together with the sequence on 5 -end were necessary for the binding activity to a highly conserved loop between βA and βB strands of ED3. Although S15 aptamer was selected against DENV-2, the authors demonstrated that this aptamer can neutralize the infections by all four serotypes of DENVs [182] .
The DENV capsid (C) protein functions as a structural component of the infectious virion but it may also have additional functions in the virus replicative cycle [183] . Balinsky et al. showed that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL) and that this interaction can be disrupted by the addition of a NCL binding aptamer (AS1411), developed as AGRO100 by Aptamera (Louisville, KY, USA). Treatment of cells with AS1411 produced a significant reduction of viral titers after DENV infection. Moreover, the authors showed that treatment with AS1411 affected the migration characteristics of the viral capsid and identified a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics [184] .

Aptamers to Ebola Virus (EV)

Ebola virus belong to the genus Ebolavirus. Four of five known viruses in this genus cause a severe and often fatal hemorrhagic fever in humans and other mammals, known as Ebola virus disease (EVD). Ebola virus has caused the majority of human deaths from EVD, and is the cause of the 2013-2015 Ebola virus epidemic in West Africa.
In many cases, aptamers have been used as a technological and research tool to identify RNA sequences that are recognized by different virus proteins. The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of Moloney murine leukemia virus (MLV), Sindbis virus (SIN) and Ebola virus [187] , by targeting the viral mRNAs for degradation and preventing the accumulation in the cytoplasm. With the aim to identify RNA sequences that could be a target of ZAP, Huang et al. used aptamer technology identifying G-rich RNA aptamers that contained conserved "GGGUGG" and "GAGGG" motifs in the loop region. Interestingly, overexpression of the aptamers significantly reduced ZAP's antiviral activity and the substitution of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity, suggesting that the RNA sequence is important for specific interaction between ZAP and the target RNA [188] .

Aptamers to Severe Acute Respiratory Syndrome (SARS)

Severe acute respiratory syndrome (SARS) is a disease caused by SARS coronavirus (SARS-CoV) that caused a pandemic pneumonia in 2002-2003, with a total of 8096 reported cases, including 774 deaths in 27 countries. SARS-CoV belongs to the Coronavirus genus in the Coronaviridae family and is an enveloped, positive-sense RNA virus with a genome of 27.9 kilobases. This RNA encodes two large polyproteins, pp1a and pp1ab, and four structural proteins including spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. Pp1a and pp1ab are proteolytically cleaved into 16 non-structural proteins (nsps) that form the viral replicase-transcriptase complex (reviewed in [189] ).
Only a few studies have been focused on the development of aptamers against SARS-CoV as antivirals, in spite of the fact that there is still no effective therapeutic treatment against the virus. Two studies have selected DNA and RNA aptamers against the non-structural nsp13 protein. This protein possesses NTPase, duplex RNA/DNA-unwinding and RNA-capping activities that are essential for viral replication and proliferation. These aptamers inhibited helicase activity with subnanomolar IC 50 , while the ATPase activity was not affected, suggesting that the aptamers may bind to the nucleic acid binding site of the helicase and block the unwinding activity [192, 193] . Table 7 shows information on the aptamers described against emerging viruses. [190, 191] Severe acute respiratory syndrome n.d. DNA/RNA non-structural nsp13 protein [192, 193] n.d. = not determined.

Perspectives

The success of treatment in viral diseases depends on the early detection of the infective agent. The most probable use of aptamers in virus diagnostics involves the development of more simple, fast and cheap diagnostics devices. One of these simple detection systems can be the Lateral Flow Immunoassays (LFIAs) which are currently used for qualitative monitoring in resource-limited or non-laboratory environments. The LFIA biosensing platform mainly comprises the sample pad and test pad, which is generally composed of nitrocellulose membrane, and provides a platform for both reaction and detection where the capturing molecules are antibodies [197] . Lateral Flow biosensing platform has been developed using an aptamer against hepatitis C virus (HCV) core antigen [83] and could be applied for HIV or emerging virus detection using aptamers against specific proteins. This method allows detection of viruses in endemic or transit of human areas. Another interesting approach to obtaining cheaper diagnostics/genotyping devices is using only one aptamer to detect several targets. From this point of view, the strategy by Wang et al. [146] , in which they use the conformational change of one aptamer exposed to different ion concentrations to detect multiple types of the influenza virus could be used for genotyping of other viruses such as HBV or HCV.
From a therapeutic point of view, aptamers offer a hopeful solution in viral diseases because they can target elements of the virus or the infected host cell easier than the antibodies mainly due to their small size. The potential design of aptamers working against different targets might block the virion penetration into the cells or inhibit enzymes responsible for viral replication or other critical processes. In the case of HIV-1, despite efficient antiretroviral therapy, eradication of latent HIV-1 provirus is challenging and requires novel biological insights and therapeutic strategies. For this aim, novel target proteins should be chosen in HIV reservoir organs for the isolation of aptamers that could be applied to drug delivery or targeting of nanoparticles loaded with drugs to obtain HIV transcriptional activation.
As already mentioned above, RIG-I has been used as a target for aptamers to control viral infection [169] . Recently, Olagnier et al. investigated the inhibitory effect of a RIG-I agonist on the replication of Dengue and Chikungunya viruses [198] . The authors demonstrated that RIG-I stimulation generated a protective antiviral response against both pathogens. It would be motivating to study the use of RIG-I aptamers developed by Hwang for Dengue and Chikungunya therapy. Likewise, the effect of the aptamers developed by Guerra et al. against PABP in decreasing replication of influenza virus [174, 199] could be studied in other viruses that also use the PABP of the infected cell [200] .

Aptamers to Reverse Transcriptase (RT)

Reverse transcriptase has two enzymatic activities, a DNA polymerase activity that can copy either a DNA or an RNA template, and an RNase H that cleaves RNA only if the RNA is part of an RNA/DNA duplex. The two enzymatic functions of RT, polymerase and RNase H, cooperate to convert the RNA into a double-stranded linear DNA [40] . DeStefano and Nair confirmed in vitro effectiveness of DNA aptamer, named 37NT, directed against the reverse transcriptase of HIV HXB2 strain. The aptamer competed with the natural template for the binding site in the enzyme, subsequently producing inhibition of the viral replication [41] . In parallel, Michalowski et al. identified three aptamers (RT5, RT6 and RT47) which contained a bimodular structure comprising a 5 -stem-loop module linked to a 3 -G-quadruplex. In addition, the authors demonstrated that this DNA aptamers inhibited RT from diverse primate lentiviruses with low nM IC 50 values [42] .

Aptamers to CCR5

HIV-1 commonly uses C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR-4) as co-receptors along with CD4 to enter target cells. Human CCR5 is an important co-receptor for macrophage-tropic virus expressed by T-cells and macrophages. Differences in CCR5 are associated with resistance or susceptibility to HIV-1. As an essential factor for viral entry, CCR5 has represented an attractive cellular target for the treatment of HIV-1. Thus, Zhou et al. have reported the selection of RNA aptamers against CCR5 using high throughput sequencing (HTS) to analyze the RNA pools from selection rounds 5 to 9. The individual sequences were classified into six major groups (Group 1-6). Group 2, 4 and 5 shared a conserved sequence, which is comprised of 10 nucleotides UUCGUCUG(U/G)G, named G3. The G3 activity was studied by a "prophylactic" HIV-1 experiment determining whether the aptamer would block HIV infectivity of R5 viruses in cell culture. The results showed that the G3 aptamer efficiently neutralized HIV-1 infectivity of R5 strains with IC 50 about 170~350 nM [25] .

Aptamers HBV in Diagnostics

One of the current objectives in the diagnostic of HBV is to develop a daily screening assay with a short period of detection between infection and recognition. Therefore, Suh et al. have developed a fast and low cost detection test based on competitive binding assay combined with fluorescence resonance energy transfer (FRET) [73] . The assay was built with an aptamer selected against the hepatitis B virus surface antigen (HBsAg), the best characterize and most frequently used HBV marker [74] . The described aptasensor was approximately 40-fold more sensitive than the conventional method. In 2015, a new set of three different DNA aptamers was selected against HBsAg and applied to develop a chemiluminescence platform. The new aptasensor was designed with aptamers-conjugated to magnetic nanoparticles reaching a detection limit five-fold better than the current enzyme-linked immunosorbent assay (ELISA) kits used in hospitals [75] .

Aptamers HCV in Diagnostics

Aptamer-based biosensors are a promising diagnostic platform to allow HCV infection detection in early stages or in immunosuppressed patients. Thus, different groups have developed diverse aptasensors to improve diagnostic assay of HCV infection. First, Lee et al. developed a biosensor prototype that specifically recognizes the HCV core protein from sera of an infected patient using selected RNA aptamers against core antigen. The HCV viral particles were retained by the 2 -F aptamers immobilized in a 96-well plate and detected by sequential steps with anti-core and Cy3-labeled secondary antibodies [80] . Later on, Chen et al. developed an early diagnostic assay based on sandwich-ELISA to recognize HCV viral proteins using biotin-labelled DNA aptamers against HCV Envelope glycoprotein E2. The obtained results from infected patients showed a good correlation between viral genome quantification assay, HCV antibody detection and sandwich-ELISA aptasensor [81] . Afterwards, Shi et al. developed a similar platform for early detection, coating the bottom of the well with C7, a DNA aptamer against HCV core labelled with biotin, and HCV-core antibody conjugated with horseradish peroxidase (HRP) is applied over the surface. The platform was applied to the detection of the protein in sera from HCV-infected patients and showed a proportional relationship between amplified RNA copies and HCV core protein concentration [82] . Moreover, Wang et al. designed a rapid, easy-to-use diagnostic platform composed of lateral flow strips treated with thiol-DNA aptamers against HCV core antigen. HCV ELISA assay and core aptamer lateral flow strips showed positive coincidence rates when compared with HCV RNA amplification assay [83] . In an effort to develop a diagnostic test to monitor the infectivity of HCV samples, Park et al. have designed an ELISA-like assay replacing the capture and detection antibodies for DNA aptamers selected against HCV E2. The Enzyme Linked Apto-Sorbent Assay (ELASA) has been described to be used for qualitative and quantitative analysis of virus in infected samples [84] . Further, two laboratories have developed label-free aptasensor to eliminate the labelling step and simplify the HCV detection method. Hwang et al. have described a highly sensitive label-free aptasensor based on nanomechanical microcantilevers. The biosensors were able to measure the surface stress due to the interaction between immobilized RNA aptamers and the HCV helicase [85] . On the other hand, Roh et al. have developed a label-free diagnostic platform to detect and quantify the presence of HCV polymerase NS5B viral protein using conjugated streptavidin-biotin RNA aptamers on an Octet biosensor [86] .

Aptamers to HCV as Antiviral Agents

Eradication of HCV disease is one of the main objectives of global public health. Currently, HCV infected patients are treated combining protease inhibitors, as Telaprevir (TVR) and Boceprevir (BOC), with pegylated-interferon and Ribavirin. However, the new direct-acting antivirals (DAA), TVR and BOC, generate a high rate of side effects, are too expensive and are also susceptible to new resistant viruses [87] . Therefore, it is necessary to develop new DAA treatments that are more effective and with fewer side effects than current therapies. To this end, different advances have been made based on aptamers against HCV and host cell proteins as therapy [88] (Figure 3 ). amplification assay [83] . In an effort to develop a diagnostic test to monitor the infectivity of HCV samples, Park et al. have designed an ELISA-like assay replacing the capture and detection antibodies for DNA aptamers selected against HCV E2. The Enzyme Linked Apto-Sorbent Assay (ELASA) has been described to be used for qualitative and quantitative analysis of virus in infected samples [84] . Further, two laboratories have developed label-free aptasensor to eliminate the labelling step and simplify the HCV detection method. Hwang et al. have described a highly sensitive label-free aptasensor based on nanomechanical microcantilevers. The biosensors were able to measure the surface stress due to the interaction between immobilized RNA aptamers and the HCV helicase [85] .
Eradication of HCV disease is one of the main objectives of global public health. Currently, HCV infected patients are treated combining protease inhibitors, as Telaprevir (TVR) and Boceprevir (BOC), with pegylated-interferon and Ribavirin. However, the new direct-acting antivirals (DAA), TVR and BOC, generate a high rate of side effects, are too expensive and are also susceptible to new resistant viruses [87] . Therefore, it is necessary to develop new DAA treatments that are more effective and with fewer side effects than current therapies. To this end, different advances have been made based on aptamers against HCV and host cell proteins as therapy [88] (Figure 3) .

Aptamers to 5 and 3 Untranslated Regions (5 and 3 UTR)

In 2003, Toulmé et al. decided to use subdomain IIId of IRES element as an antiviral target. They selected RNA aptamer and verified that isolated aptamers inhibit HCV translation in vitro and in cell culture [89] . In the same year, Kikuchi et al. isolated RNA aptamers capable of binding to the domain II of HCV IRES and showed that IRES-mediated in vitro translation was reduced from 20% to 40% by using the 2-02 aptamer [90] . Later, they isolated a new RNA aptamer population against the HCV IRES domains III-IV and corroborated that 3-07 aptamer had a high inhibitory effect on IRES-mediated translation in vitro and in vivo [91] . To improve the inhibitory effect of selected aptamers, they constructed two new molecules, named 0207 and 0702, composed by 2-02 and 3-07 aptamers linked by their ends. The fused aptamers recognized two different subdomains of IRES element and are at least 10 times more efficient than the parental aptamers in the inhibition of mRNA IRES-dependent translation in vitro [92] . Following with IRES as an anti-viral target, Romero-López et al. described an innovative in vitro selection method to isolate aptamers fused to a hammerhead ribozyme with capacity to inhibit RNA translation mediated by IRES. Selected chimeric aptamer-ribozymes were able to recognize the IRES element and cleavage the 5 end at nucleotide position 363 [93] . The success of combining two functional elements in the same molecule was shown in the selected chimeric molecule HH363-50. Thus, the aptamer-ribozyme chimera did anchor to domain IV of the IRES element and inhibited in vitro and in vivo IRES-mediated translation [94] . Therefore, recruitment of ribosomal particles mediated by the IRES element was inhibited by the chimera HH363-24 that prevented both translation and replication in a hepatic cell line [95] . Moreover, to avoid HCV genome replication, Konno et al. isolated RNA aptamers against the 3 end of the negative strand of the virus genome [96, 97] . Interestingly, a RNA aptamer, named AP30, was able to recognize this minus-IRES region and reduce positive-strand genomic RNA synthesis [96] . To inhibit HCV replication, Marton et al. selected RNA aptamers against CRE element that were able to repress replication of HCV replicon in hepatic cells [98] . Subsequently, two selected aptamers, P58 and P78, interact with subdomain 5BSL3.2 of the CRE element and produce a structural reorganization of the 3 end HCV genome and a significant decrease of HCV replication in vivo [99] .

Aptamers to Nonstructural Protein 5A (NS5A)

It has been reported that NS5A protein is essential for HCV production and replication. Recently, Yu et al. have isolated and characterized DNA aptamers against HCV NS5A protein. Particularly, selected aptamer NS5A-5 was able to inhibit HCV virus infection by prevention of protein-protein interactions between NS5A and core protein [110] .
Aptamers to Nonstructural Protein 5B (NS5B) HCV nonstructural protein 5B (NS5B) is a RNA-dependent RNA polymerase protein (RdRp) responsible to the generation of positive-sense genomic HCV RNA and negative-sense RNA template. Reduction of HCV NS5B polymerase activity affects HCV viral life cycle and is one of the main objectives to isolate aptamers against NS5B. Thus, Biroccio et al. identified specific RNA aptamers against a truncated protein NS5B-∆55 without the C-terminal region. One of the selected aptamers, B.2, blocked RNA transcription but not competed with the complex RdRp-RNA, using different binding site than RNA template to the NS5B protein [111] . In the same way, Bellecave et al. selected DNA aptamers against the NS5B viral protein. One of the chosen aptamers, 27v, competed with positive and negative sense HCV viral RNA to bind RdRp polymerase and blocked initiation and elongation steps of RNA transcription [112] . However, 127v aptamer partially competes to dissociate RdRp-RNA complex formation and only inhibited initiation steps of HCV transcription [113] . Moreover, interference of viral production and transcription inhibition of HCV virus was confirmed in vivo using 27v aptamer. Table 4 shows information on the aptamers described against HCV. Five years later, another set of RNA aptamers against NS5B protein were obtained by Lee et al. [114] . To avoid aptamer degradation, oligonucleotides were modified with 2 hydroxyl (R-OH) or fluoropyrimidines (R-F). The R-OH aptamers blocked RNA synthesis of HCV replicon in cell culture without emergence of virus escape mutant or cellular toxicity. On the other hand, R-F oligonucleotides were truncated and conjugated with cholesterol-or galactose-PEG molecules to allow direct and specific liver delivery into cells or tissue. Cholesterol-and Gal-PEG-R-F t2 conjugated aptamer blocked RNA synthesis of HCV genome [115] . The above mentioned aptamers were non-genotype-specific; however, Jones et al. described for the first time aptamers against NS5 protein that exclusively recognized and inhibited RNA-polymerase activity of HCV virus subtype 3a [116] .

Aptamers to Structural Proteins E1, E2 and Core

Envelope E1 and E2 glycoproteins are putative targets in therapy due to its role in HCV viral recognition to enter into hepatic cells. Chen et al. have isolated DNA aptamer against E2 glycoprotein. The selected aptamers have higher affinity to genotype 1a, 1b and 2a than others, and strongly prevented HCV viral infection in Huh7 5.1 cells [81] . Afterwards, Yang et al. described the potential antiviral action of DNA aptamers selected against E1E2 protein by HCV infection suppression in HuH7.5 cells without innate immune response action [117] . In the case of core, an essential protein for HCV viral assembly, Shi et al. have applied for therapy the above-mentioned aptamers in diagnostics. In Huh7.5 cells, the aptamers against HCV core protein repressed viral production as a result of defective assembly of virus particle without stimulation of innate immune response [82] .

Aptamers to Influenza Virus in Diagnostics

The antibodies are the most common probe used to detect either the viral particles or host antibodies developed during the infection. However, although in most cases antibodies are able to distinguish between influenza A and B, only a few antibodies that differentiate subtypes of Influenza A or B have been reported. Alternative probes for subtyping are the aptamers. Thus, aptamers to hemagglutinin (HA) have been successfully and broadly used for the development on sensors for influenza detection. HA is expressed in high amounts in the viral surface and is required for binding and fusion with the host cell. Currently, more than 40 DNA and RNA aptamers to HA have been described since 2004, selected to recombinant hemagglutinins (H1, H3, H5, H9 and Ha from virus B) and to whole viruses (H5N1) (reviewed in [138] ). Misono and Kumar selected an RNA aptamer against to HA of A/Panama/ 2007/1999 (H3N2) using SPR-based SELEX [139] . Gopinath et al. generated two RNA aptamers against intact influenza virus A/Panama/2007/1999 and HA of B/Johannesburg/05/1999. These RNA aptamers are able to discriminate among both A and B influenza viruses [140, 141] .

Aptamers to Influenza Virus as Antiviral Agents

Other aptamers targeting NS1 or the PA polymerase subunit 1 have also been studied. NS1 is a nonstructural protein of small size, between amino acids 230 and 238 and with a molecular weight of 26 kDa. In view of its interaction with both RNAs and viral and cellular proteins, NS1 has been implicated in many of the alterations that occur during influenza virus infection. Moreover, NS1 has anti-interferon (IFN) properties leading to the inhibition of the host's innate immunity [162] . Thus, the importance of NS1 in viral infection makes it an attractive therapeutic target. Woo et al. selected a DNA aptamer specific to NS1 that induced IFN-β production by inhibiting NS1 function. In addition, the selected aptamer was able to inhibit the viral replication without affecting cell viability [163] .

Aptamers to Dengue Virus (DENV)

From a technological point of view, aptamers have been used for efficient isolation of endogenously assembled viral RNA-protein complexes. Hence, Dong et al. developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs) using the streptavidin-binding aptamer S1 sequence that was inserted into the 3 end of dengue virus (DENV) 5 -3 UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. This strategy led to identify several novel host DENV RBPs by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which were further implicated in DENV replication [185] .

Aptamers to Ebola Virus (EV)

Viral protein 35 (VP35) is a multifunctional dsRNA binding protein that plays important roles in viral replication, innate immune evasion and pathogenesis. These multifunctional proteins offer opportunities to develop molecules that target distinct functional regions. With this purpose, Binning et al. used a combination of structural and functional data to determine regions of Ebola virus (EBOV) VP35 (eVP35) to target aptamer selection. Two distinct classes of aptamers were characterized based on their interaction properties to eVP35. These results revealed that the aptamers bind to distinct regions of eVP35 with high affinity (10-50 nM) and specificity. In addition, the authors showed that these aptamers compete with dsRNA for binding to eVP35 and disturb the eVP35-nucleoprotein (NP) interaction. Consistent with the ability to antagonize eVP35-NP interaction, select aptamers can inhibit the function of the EBOV polymerase complex reconstituted by expression of select viral proteins [186] .

Aptamers to HIV as Antiviral Agents

At present, the treatment HIV-1/AIDS is by a combination of several antiretroviral drugs (cART), which can slow the progress of the disease and reduce the risk of death and disease complication, but it is not curative. Moreover, many patients do not tolerate cART because it has severe side effects, and it is too expensive for patients in developing countries. In this regard, aptamers have been considered an alternative or adjuvant to the chemical antiviral agents in cART to overcome these limitations. To date, highly specific, nucleic acid-based aptamers that target various parts of HIV-1 genomes, HIV-1 proteins (including HIV-1 protease (PR), reverse transcriptase (RT), nucleocapsid, gp120, and Gag) and cellular proteins (nucleolin, CD4 or CCR5) have been isolated and shown to effectively suppress viral replication to apply in HIV therapy [5, [25] [26] [27] (Figure 2 ). diamond field-effect transistor (FET) technique [24] . These "apta-biosensors" have high sensibility and specificity but the devices are complex and expensive.

Perspectives

In the field of viral diseases, the number of drugs for treating these infections is very small and most of the available therapeutics are not very effective [194] . In addition, current diagnostic tools for viral infections are expensive and time consuming. These important diagnostic and therapeutic limitations have favored the development of aptamer-based systems, mainly because these show several interesting advantages in relation to antibodies. Thus, aptamers can recognize and/or inhibit target activity through specific and strong interactions superior to other biologics and small molecule therapeutics, with lower toxicity and immunogenicity profiles. In this sense, during the last years, aptamer technology is being used in a wide range of diagnostic and therapeutic applications associated with viral pathologies [195, 196] . It is significant that aptamers selected as specific anti-viral molecules are effective in infected cells, however, none of the selected antiviral aptamers entered into clinical trials. In conclusion, it is necessary to continue research studies and successfully develop clinical trials to establish the use of aptamers as antivirals.
Nanoparticles have been considered for a wide range of applications, both in soluble and insoluble forms. An advantage of soluble forms of nanoparticles is that they can encapsulate antibiotics/drugs and then release them when they reach the cellular environment, making them highly applicable for drug delivery systems. Studies on aptamer-based enhanced drug delivery have been reported for prostate cancer and lymphoblastic leukemia cells [201] . With this purpose, for HIV therapy, nanoparticles successfully loaded with the antiretroviral (ARV) drugs efficiently inhibited HIV-1 infection [202, 203] . These results showed the benefit of the nanoparticles' application to delivery of antiviral drugs to improve its bioavailability.
In conclusion, the use of aptamers in the development of diagnostic platforms or as therapeutic drugs is a promising alternative for the treatment of viral diseases.
20 section matches

Abstract

The flavivirus Zika virus (ZIKV) activates ribonuclease L (RNase L) catalytic antiviral 26 function during infection, yet deletion of RNase L decreases ZIKV production, suggesting 27 a proviral role of RNase L. In this study, we reveal that latent RNase L supports ZIKV 28 replication factory (RF) assembly. Deletion of RNase L induced broader cellular 29 distribution of ZIKV dsRNA and NS3 compared with densely concentrated RFs detected 30 in WT cells. An inactive form of RNase L was sufficient to contain ZIKV genome and 31 dsRNA within a smaller area, which increased levels of viral RNA within RFs as well as 32 infectious ZIKV released from the cell. We used a microtubule stabilization drug to 33 demonstrate that RNase L deletion impaired the cytoskeleton rearrangements that are 34 required for proper generation of RFs. During infection with dengue or West Nile Kunjin 35 viruses, RNase L decreased virus production, suggesting that RNase L proviral function 36 is specific to ZIKV. 37 38 39 Keywords 40 Zika virus, flavivirus, RNase L, replication factories, dengue virus, West Nile Kunjin virus 41 42 43 44 45 46 47 All rights reserved. No reuse allowed without permission.

Summary 25

The flavivirus Zika virus (ZIKV) activates ribonuclease L (RNase L) catalytic antiviral 26 function during infection, yet deletion of RNase L decreases ZIKV production, suggesting 27 a proviral role of RNase L. In this study, we reveal that latent RNase L supports ZIKV 28 replication factory (RF) assembly. Deletion of RNase L induced broader cellular 29 distribution of ZIKV dsRNA and NS3 compared with densely concentrated RFs detected 30 in WT cells. An inactive form of RNase L was sufficient to contain ZIKV genome and 31 dsRNA within a smaller area, which increased levels of viral RNA within RFs as well as 32 infectious ZIKV released from the cell. We used a microtubule stabilization drug to 33 demonstrate that RNase L deletion impaired the cytoskeleton rearrangements that are 34 required for proper generation of RFs. During infection with dengue or West Nile Kunjin 35 viruses, RNase L decreased virus production, suggesting that RNase L proviral function 36 is specific to ZIKV. report of viral resistance to catalytically active RNase L during infection, we sought to 113 isolate the differences between ZIKV RFs and those constructed by other flaviviruses, to 114 identify factors that enable this ZIKV evasion mechanism. 115 All rights reserved. No reuse allowed without permission.
Since it is well understood that flaviviruses reorganize the cellular cytoskeletal and 119 organellar network during infection (Neufeldt et al., 2018) , we investigated the possibility 120 that RNase L association with the cytoskeleton was exploited by ZIKV to assemble highly 121

Results 125

RNase L-mediated cleavage of rRNA ( Figure 1B) . In contrast to other RNase L-activating 133 viruses, RNase L nuclease activity did not restrict ZIKV production. This was 134 demonstrated by a lack viral titer increase by 48hpi, after multiple rounds of replication, 135 in ZIKV-infected RNase L KO cells compared to in WT cells ( Figure 1C) . Instead, deletion 136 of RNase L reduced ZIKV production. In comparison to ZIKV, we detected weaker 137 activation of RNase L by West Nile virus Kunjin strain (KUNV), although also OAS3-138 All rights reserved. No reuse allowed without permission.
Since infectious virus production was enhanced by RNase L expression, we examined 146 whether RNase L was required for optimal ZIKV replication. We used 147 immunofluorescence assays (IFAs) for detection of the replication intermediate dsRNA 148
RNase L-mediated rRNA degradation, which was only detectable in ZIKV infected +RL 312 WT cells and not VC or +RL R667A cells, despite OAS3 protein expression in all three 313 cell lines (Figure 6A&B) . We evaluated effects of RNase L WT or R667A expression on 314 ZIKV dsRNA expression and localization compared to that of VC cells at 20hpi, using PDI 315 ER staining to denote RF sites. We found that expression of either RL WT or RL R667A 316 increased ZIKV dsRNA intensity at the ER over that in VC cells, however only RL R667A 317 increased circularity of ZIKV RFs in comparison to those of VC cells (Figure 6C&D) . To 318 determine if RNase L effects on ZIKV dsRNA intensity or circularity correlated with 319 elevated infectious ZIKV titers, we measured virus from infected HeLa M cells at 48hpi 320 and detected a significant increase in ZIKV production in RL R667A-expressing cells 321 compared to VC or RL WT-expressing cells (Figure 6E ). In addition, we found that neither 322 All rights reserved. No reuse allowed without permission.
Furthermore, expression of RL WT, but not RL R667A, slightly decreased KUNV 325 production at 48hpi ( Figure 6E) . As RNase L is activated during KUNV infection at this 326 timepoint, restricted virus production is likely a result of RNase L catalytic activity to inhibit 327 KUNV replication. RFs, although the mechanism by which paclitaxel inhibits ZIKV infection has not been 343

Paclitaxel treatment of both ZIKV infected WT and RNase L KO cells resulted in btubulin 365

aggregation due to inhibition of microtubule tractability ( Figure 7E) . Finally, we treated 366 cells with paclitaxel 3h after infecting with ZIKV to measure changes in infectious virus 367 production at 24hpi as a result of microtubule stabilization in WT and RNase L KO cells. 368 All rights reserved. No reuse allowed without permission.

Discussion 377

In particular, a catalytically inactive form of RNase L improved ZIKV RF assembly and 383 function, thereby boosting virus production. We also provide evidence suggesting that 384 ZIKV repurposes the interaction between RNase L and the cytoskeleton to facilitate 385 rearrangement of the ER for establishment of ZIKV RFs. While cytoskeletal arrangements 386 for RF assembly within ER folds are characteristic of flaviviruses, we found that RNase L 387 expression during DENV and KUNV infection had only minimal effects on RF formation. 388
Furthermore, RNase L was ultimately restrictive of both viruses, operating in its canonical 389 antiviral manner to decrease DENV and KUNV titers through its catalytic activity. 390 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/852194 doi: bioRxiv preprint Therefore, we propose that recruitment of RNase L for enhanced RF function and virus 391 release is a feature presently observed only during ZIKV infection. 392
Supernatants from infected cells was harvested at 2 or 24hpi for measurement of viral 551 titers over time by plaque assay, shown as plaque forming units (PFU)/mL virus. Data is 552 representative of at least two independent experiments. Statistical significance was 553 determined by one-way ANOVA, displayed is comparison of WT ± pIC (****), and the 554 mean of three replicates ± SD, ****p<0.0001. Imaged at 60X magnification, scale bar 555 500µm. 556 representative of at least two independent experiments. Statistical significance was 571 determined by one-way ANOVA, displayed is the mean of three replicates ± SD for 572 All rights reserved. No reuse allowed without permission.

Virus replication assays 606

Cells were seeded into 24-well plates and infected the next day at indicated MOI in 607 triplicate. After 1h incubation of cells with virus at 37°C, virus was removed and cells 608 washed three times with PBS before addition of 1mL complete media. At indicated time 609 post-infection, 150µl supernatants were harvested and stored at -80°C until titration, and 610 150µl complete media replaced in each well so that all time points from one replicate 611 contain supernatant from the same infected well throughout the time course. Data is 612

Plaque assays 614

Virus supernatant from infected cells was serially diluted in DMEM supplemented with 2% 615 FBS and added to Vero cell monolayers in 6-well plates. Plates were incubated for 1 hour 616 at 37°C before overlaying infected monolayers with DMEM containing 3% FBS, 8% 617 NaCO3, 10mM HEPES, 1X L-glutamine, 250µg/mL amphotericin B, and 0.7% agarose. 618 All rights reserved. No reuse allowed without permission.

Results 125

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/852194 doi: bioRxiv preprint dependent, and could not detect any RNase L activation by DENV using this assay 139 ( Figure 1B) . Despite reduced RNase L activation by DENV and KUNV, both viruses were 140 restricted by RNase L activity in WT cells ( Figure 1C) . Therefore, RNase L is not only 141 ineffective in inhibiting ZIKV infection, but also specifically promotes ZIKV production 142 ( Figure 1A, right panel) . 143 144 RNase L improves ZIKV RF function to increase ZIKV RNA and protein expression. 145
and RNase L KO cells. At 20hpi, expression of both ZIKV dsRNA and NS3 was increased 150 in WT cells, as measured by mean dsRNA and NS3 intensity at perinuclear ER sites 151 characteristic of flaviviruses ( Figure 2AB&C) . We also observed a change in localization 152 of ZIKV dsRNA and NS3 in RNase L KO cells, both of which were more disseminated 153 around the nucleus and into the cytoplasm. We used circularity and diameter parameters 154 to quantify the spread of viral products throughout the cell at 20hpi, and determined that 155
to that of WT cells, we found that dsRNA intensity and circularity were decreased while 165 diameter was increased without RNase L, a similar trend but to a lesser degree than with 166 ZIKV ( Figure 2D&F) . Furthermore, this does not result in a defect in virus release as 167 measured by viral titer, but instead virus titer is enhanced by RNase L KO (Figure 1C The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. We also found that by 48hpi OAS3 KO, which suppresses RNase L activation, 201 restored infectious ZIKV production from reduced levels of RNase L KO cells to that 202 observed during WT cell infection ( Figure 3D ). DENV production, which was limited by 203
RNase L mediated antiviral activity ( Figure 3D ). These results from cells lacking OAS3 209 suggest that ZIKV infection may utilize an inactive form of RNase L in a unique manner 210 that results in higher virus production than with RNase L absent. revealed that deletion of RNase L did not alter intensity of genome expression when 230 All rights reserved. No reuse allowed without permission.

Discussion 377

In this study, we present findings supporting a proviral role of the host RNase L protein, 378 which has otherwise been considered strictly antiviral. RNase L nucleolytic activity is 379 highly effective at restricting replication of a diverse range of DNA and RNA viruses 380 (Silverman, 2007) . However, catalytic activation of RNase L by ZIKV does not limit 381 infection, and instead ZIKV titers are paradoxically increased with RNase L expression. 382
Interestingly, DENV production in WT cells was limited by RNase L, as titers 393 increased significantly in RNase L KO cells ( Figure 1C ), although we were unable to 394 detect any rRNA degradation in DENV-infected WT cells ( Figure 1B btubulin, to less concentrated areas that generated less virus, we hypothesized that this 457 was due to a loss of cytoskeletal remodeling without RNase L. Although we attempted to 458 All rights reserved. No reuse allowed without permission.
15 section matches

Abstract

Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.
The unprecedented magnitude and scope of the catastrophic 2014-2015 EBOV outbreak in West Africa, and its continued global emergence underscores the urgent need to better understand the biology and pathogenesis of this zoonotic pathogen. We have identified BAG3 as a novel and functional host VP40 interactor that negatively regulates VP40 VLP and virus egress in a PPxY/WW-domain dependent manner. As a cell survival protein and key regulator of chaperone-mediated autophagy (CMA), BAG3 sequesters EBOV and MARV VP40 away from the site of budding at the plasma membrane, and thus may represent a novel host defense strategy to combat filovirus VP40-mediated egress and spread.

The filovirus matrix protein VP40 is the most abundant protein in the virion and is essential for virus assembly and egress. Indeed, expression of VP40 alone is sufficient to form viruslike particles (VLPs), which are morphologically indistinguishable from infectious virions and are released from mammalian cells in a manner that recapitulates the release of authentic virus [2] [3] [4] [5] [6] . Although not required for EBOV replication [7] , Late (L) domains (which contain PTAP and/or PPxY amino acid sequence motifs) are conserved within EBOV and MARV VP40 and promote efficient egress of VLPs and virus by recruiting host proteins that facilitate virus-cell separation [3, 4, 6, [8] [9] [10] [11] . For example, EBOV and MARV VP40 L-domains hijack specific host proteins associated with the ESCRT pathway, including Tsg101, Alix, and Nedd4 [3, 6, [8] [9] [10] [11] [12] [13] .

BAG3 inhibits egress of infectious recombinant virus VSV-M40

Finally, we sought to determine whether expression of BAG3 would inhibit egress of infectious virus. Toward this end, we utilized our live infectious VSV recombinants; VSV-M40 and VSV-M40-P2728A [10] . Recombinant VSV-M40 expresses the WT L-domain motifs (PTAP-PEY) and flanking residues from eVP40 in place of the L-domain of VSV M protein and buds efficiently, whereas recombinant VSV-M40-P2728A expresses mutated eVP40 L-domain motifs (PTAAAEY) and is budding defective [10] . Briefly, HEK293T cells were first transfected with vector alone, BAG3-WT, or BAG3-ΔN for 24 hours, and then infected with either VSV-M40 or VSV-M40-P2728A at an MOI of 0.1 for 8 hours (peak time of budding). Infected cell extracts were analyzed by Western blot for expression controls, and virus production was quantified by standard plaque assay (Fig 10) . We found that the levels of infectious VSV-M40 released from mock-and BAG3-ΔN-transfected cells were virtually identical; however, titers of VSV-M40 released from cells expressing BAG3-WT were consistently and significantly reduced by >50% compared to controls (Fig 10A) . In contrast, expression of BAG3-WT did not have any significant effect on budding of mutant VSV-M40-P2728A (Fig 10C) . Importantly, these data demonstrate that the inhibitory effect of BAG3 on budding extends to infectious virus, and confirms the involvement of the viral PPxY/BAG3 WW-domain interaction in this negative regulatory mechanism.

Cytosol and plasma membrane protein fractionation

Transfection/Infection assays HEK293T cells were first transfected with BAG3, BAG3-ΔN or vector for 24 hours, and then subsequently infected with either VSV-M40 or VSV-M40-P2728A at a MOI of 0.1. Supernatants and infected cell extracts were harvested at 8 hours post-infection. Released VSV-M40 and VSV-M40-P2728A virions were titrated in duplicate via standard plaque assay on BHK-21 cells. Cellular proteins were detected by Western blotting using specific antibodies.

Introduction Ebola (EBOV) and Marburg (MARV) viruses are virulent pathogens that cause severe hemorrhagic disease in humans and non-human primates. There are currently no FDA approved vaccines or antiviral drugs to prevent or treat infections by these Category A NIAID priority pathogens [1] . The recent catastrophic outbreak of EBOV in West Africa underscores the urgent need to better understand the biology and pathogenesis of this global public health threat, and to decipher the molecular mechanisms by which EBOV interacts with the host to cause disease.

Discussion

As the major filovirus matrix protein, VP40 plays a central role in directing virion assembly and egress from infected cells. Three minimal functional domains of VP40 are required for efficient VLP egress, including a membrane (M) binding region, a self-interaction (I) domain, and one or more late (L) domain motifs. The L-domain motifs hijack or recruit specific host cell proteins that facilitate or promote efficient virus-cell separation [3, 4, 6, [48] [49] [50] [51] [52] [53] . Here, we

Fig 8. BAG3 alters the intracellular localization of eVP40 in live cells. A)

doi:10.1371/journal.ppat.1006132.g009 BAG3 is the only member of this family that contains a single N-terminal WW-domain. As a co-chaperone and cell survival protein, BAG3 regulates multiple cell pathways, including, apoptosis, autophagy, cell development and cytoskeleton organization [45, 55, 56] . Indeed, BAG3 is induced under conditions of cell stress and plays a major role in sequestering misfolded and/ or foreign proteins to the proteasome for degradation by CMA [45, 55, 56] . Our data imply that the CMA function of BAG3 acts as a novel host defense/response mechanism to sequester a fraction of VP40 from the site of budding at the PM and into aggresomes, thus reducing VLP/ virus egress and spread. Whether sequestration of VP40 away from the PM results in degradation of VP40 remains to be determined. Interestingly, selective autophagy of the endoplasmic reticulum (ER-phagy) was recently shown to regulate EBOV replication in murine cells [57] .
BAG3 has been associated with the lifecycles of other RNA and DNA viruses including, HIV-1, varicella zoster virus, herpes simplex virus, African swine fever virus, papillomavirus, polyomaviruses, coronavirus, adenovirus, and Epstein Barr virus [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] . However, in contrast to our findings with VP40, BAG3 primarily exerts positive effects on the lifecycles of these other viruses. For example, Gout et. al [63] reported that the adenovirus penton base protein interacted with the WW-domain of BAG3 via its PPxY motif to promote adenovirus entry and virus progeny production.
In sum, we identified BAG3 as a novel, WW-domain interactor with the PPxY motif of VP40 leading to inhibition of VLP and infectious virus egress in a PPxY/WW-domain dependent manner. As a stress-induced, cell survival protein, BAG3 may represent a key component of a novel host defense mechanism to dampen virus egress via CMA and protein sequestration. These findings provide new insights into the roles that host proteins play in regulating filovirus VP40-mediated egress, and a more comprehensive understanding of these virus-host interactions may be helpful in the design of future antiviral therapies. For example, it may be possible to identify small molecules that could bind to VP40 in a manner that mimics the inhibitory effect of BAG3. Alternatively, the WW domain of BAG3 alone (per se) could be used to inhibit VLP and virus egress as documented previously for cis-expressed YAP WW domain that inhibited PPxY-mediated budding of Rous Sarcoma virus [71] .

VLP budding assay and BAG3 titration

Filovirus VP40 VLP budding assays in HEK293T cells were described previously [2, 13, 35, 72, 73] . eVP40 and mVP40 proteins in VLPs and cell extracts were detected by SDS-PAGE and Western blotting, and quantified using NIH Image-J software. The anti-eVP40 antiserum was used to detect eVP40-WT and eVP40-ΔPT/PY mutant, and anti-flag monoclonal antibody was used to detect flag-tagged mVP40. For BAG3 titration experiments, HEK293T cells were transfected with 0.1μg of eVP40 or mVP40 and increasing amounts of BAG3-WT or BAG3-ΔC (0.1, 0.5, 1.0μg), or BAG3-ΔN (0.1 and 1.0μg). The total amounts of transfected DNA were equivalent in all samples. Supernatants and cell extracts were harvested at 24 hours post transfection.

Viral proteins bearing PPxY motif each interact with a unique repertoire of WW-domain bearing host proteins with diverse functions [14] [15] [16] [17] [18] [19] [20] [21] [22] . For example, the PPxY L-domain within eVP40, mVP40, and other viral matrix proteins interacts specifically with WW-domains of: 1) host Nedd4; a HECT family E3 ubiquitin ligase that is linked with the cellular ESCRT machinery, 2) host ITCH; a HECT family E3 ubiquitin ligase involved in immune regulation and inflammatory signaling, and 3) host IQGAP1; a multifunctional scaffolding protein involved in regulating cell motility, actin polymerization, and filopodia formation [2, [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] . In general, these previously characterized viral PPxY/WW-domain interactions promote efficient virus production.
Here, we sought to identify additional WW-domain bearing proteins that interact with the eVP40 PPxY motif by screening a GST array of 115 host proteins containing one or more WW-domains [39] with an EBOV PPxY-containing peptide. Using this unbiased approach, we identified WW-domain containing protein BAG3 as a novel eVP40 interactor. BAG3 is a stress-induced molecular co-chaperone involved in regulating cellular protein homeostasis by CMA. Since in general, viral PPxY-containing proteins tend to bind WW-domains with good specificity and selectivity [40] , our identification of BAG3 suggests that this protein may play a biologically relevant role in the lifecycle of EBOV. Indeed, we confirmed that hypothesis by first using co-IP to validate the specificity of the PPxY/WW-domain physical interaction between VP40 (both eVP40 and mVP40) and BAG3, and functionally demonstrated that expression of BAG3 inhibited VP40 VLP production, as well as budding of a VSV recombinant virus containing the EBOV VP40 PPxY L-domain motif. To our knowledge, this is the first identification of a VP40-interacting mammalian WW-domain bearing protein that negatively regulates budding. Mechanistically, our data suggest that BAG3 binds VP40 and not only sequesters it away from the site of budding at the plasma membrane, but also directs a fraction of VP40 into aggresomes, thus reducing VLP egress.

BAG3 inhibits VP40 VLP egress

To determine whether the novel inhibitory effects of BAG3 on VP40 VLP egress were dosedependent, we transfected HEK293T cells with a constant amount of eVP40 or mVP40 plus increasing amounts of BAG3-WT, BAG3-ΔN or BAG3-ΔC. Cell extracts and supernatants were harvested as described above (Fig 6) . Appropriate expression levels for all proteins were confirmed by Western blotting of cell extracts (Fig 6, Cells) . We observed a clear dose-dependent inhibition of both eVP40 (Fig 6A and 6B ) and mVP40 (Fig 6D and 6E ) VLPs in the presence of increasing amounts of either BAG3-WT or BAG3-ΔC. In contrast, increasing expression of WW-domain deletion mutant BAG3-ΔN had no effect on budding of either eVP40 or mVP40 VLPs (Fig 6C and 6F ). Once again, the level of inhibition mediated by BAG3-WT and BAG3-ΔC appeared to be more pronounced on mVP40 VLPs compared to that on eVP40 VLPs, with mVP40 VLPs being virtually undetectable in samples expressing the highest amounts of BAG3. Taken together, these results demonstrate a functional role for BAG3 as a negative regulator of eVP40 and mVP40 VLP budding via a WW-domain dependent mechanism. To our knowledge, this is the first host WW-domain containing protein shown to inhibit filovirus VP40 egress.

Fig 8. BAG3 alters the intracellular localization of eVP40 in live cells. A)

doi:10.1371/journal.ppat.1006132.g008 have identified host WW-domain containing protein BAG3 as a novel interactor with the PPxY L-domain motif of both eVP40 and mVP40. Moreover, we confirmed the physical and functional interaction between the WW-domain of BAG3 and the viral PPxY L-domain motif by using L-domain and WW-domain mutants in GST-pulldowns, co-immunoprecipitation, siRNA analysis, and/or VLP/virus budding assays. Intriguingly, unlike previously identified host WW-domain proteins that interact with VP40, including Nedd4 [2, 28, 32, 35] and ITCH [37] , BAG3 is the first host WW-domain interactor to negatively regulate egress of eVP40 and mVP40 VLPs, as well as infectious virus containing the eVP40 PPxY L-domain motif.